HMGA2 Moderately Increases Fetal Hemoglobin Expression in Human Adult Erythroblasts.

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with beta-hemoglobin disorders. Previous studies showed that let-7 microRNAs (miRNAs) are highly regulated in erythroid cells during the fetal-to-adult developmental transition, and that targeting let-7 mediated the up-regulation of HbF to greater than 30% of the total globin levels in human adult cultured erythroblasts. HMGA2 is a member of the high-mobility group A family of proteins and a validated target of the let-7 family of miRNAs. Here we investigate whether expression of HMGA2 directly regulates fetal hemoglobin in adult erythroblasts. Let-7 resistant HMGA2 expression was studied after lentiviral transduction of CD34(+) cells. The transgene was regulated by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2-OE). HMGA2-OE caused significant increases in gamma-globin mRNA expression and HbF to around 16% of the total hemoglobin levels compared to matched control transductions. Interestingly, no significant changes in KLF1, SOX6, GATA1, ZBTB7A and BCL11A mRNA levels were observed. Overall, our data suggest that expression of HMGA2, a downstream target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in adult human erythroblasts

Erythroid-expression of HMGA2 leads to slightly lower levels of terminal maturation and enucleation compared to control transductions during <i>in vitro</i> erythroblast differentiation.

<p>Representative donor flow cytometry analyses of <b>(A)</b> empty vector control and HMGA2-OE at culture days 14 and 21 stained with anti-transferrin receptor (CD71) and anti-glycophorin A (GPA) antibodies. <b>(B)</b> Enucleation of empty vector control and HMGA2-OE transductions assessed by staining of culture day 21 cells with thiazole orange. Percentages shown in the figure correspond to one representative donor, average values are shown in the text. C = empty vector control transduction; HMGA2-OE = HMGA2 over-expression.</p

Enforced expression of HMGA2 regulates <i>gamma-globin</i> mRNA and pancellular HbF levels in adult human erythroblasts cultivated <i>ex vivo</i>.

<p>Quantitation of copy number per nanogram cDNA (copies/ng cDNA) by Q-RT-PCR analyses was performed at culture day 14 for <b>(A)</b> <i>alpha-</i>, <i>mu-</i>, <i>theta-</i> and <i>zeta-globins</i>, and <b>(B)</b> <i>beta-</i>, <i>delta-</i>, <i>gamma-</i> and <i>epsilon-globins</i> in HMGA2-OE and empty vector control transductions. Representative HPLC profile of hemoglobin collected at culture day 21 from <b>(C)</b> empty vector control transductions and <b>(D)</b> HMGA2-OE. <b>(E)</b> Average percentage of HbF levels detected by HPLC analysis. <b>(F)</b> Representative flow cytometry dot plots of empty vector control transductions (left panel) and HMGA2-OE (right panel) at culture day 21 stained for fetal hemoglobin. Open bar represents empty vector control and black bar represents HMGA2-OE. Mean value ± SD of four independent donors for each condition. P-value was calculated using one-tailed paired Student’s t-test. C = empty vector control transduction; OE = HMGA2 over-expression. *p<0.05.</p

HMGA2 does not regulate the <i>let-7</i> family of miRNAs.

<p>Effects in the expression levels of the <i>let-7</i> family of miRNAs were analyzed in HMGA2-OE and empty vector control transductions. Quantitation of copy number per nanogram cDNA (copies/ng cDNA) by Q-RT-PCR analyses was performed at culture day 14. Open bars represent empty vector control and black bars represent HMGA2-OE. Mean value ± SD of four independent donors for each condition. C = empty vector control transduction; OE = HMGA2 over-expression.</p

HMGA2 expression has mild effects in erythroid- and HbF-related transcription factors.

<p>Expression level of <b>(A)</b> <i>KLF1</i>, <b>(B)</b> <i>SOX6</i>, <b>(C)</b> <i>GATA1</i>, <b>(D)</b> <i>ZBTB7A</i> and <b>(E)</b> <i>BCL11A</i> were analyzed in HMGA2-OE and empty vector control transductions. Quantitation of copy number per nanogram cDNA (copies/ng cDNA) by Q-RT-PCR analyses was performed at culture day 14. Open bars represent empty vector control and black bars represent HMGA2-OE. Mean value ± SD of four independent donors for each condition. C = empty vector control transduction; OE = HMGA2 over-expression.</p

HMGA2 over-expression was confirmed at the mRNA and protein levels.

<p><b>(A)</b> Quantitation of copy number per nanogram cDNA (copies/ng cDNA) by Q-RT-PCR in empty vector control and HMGA2-OE samples. Open bars represent empty vector control and black bars represent HMGA2-OE. Four independent donors were performed for each condition. <b>(B)</b> Western blot analysis of HMGA2 in the nuclear extracts from four independent donors upon HMGA2-OE compared to transduction controls. Blot was probed with anti-HMGA2 antibody and Lamin B1 was used as loading control. C = empty vector control transduction; OE = HMGA2 over-expression.</p

<i>LIN28A</i> over-expression mediated by KLF1 or SPTA1 promoter regulates the <i>let-7</i> family of miRNAs.

<p>RNA samples from erythroblasts cultured on day 14 were examined for <b>(A)</b><i>LIN28A</i> over-expression and <b>(B)</b> the total levels of <i>let-7</i> miRNAs using Q-RT-PCR. Mean value ± SD from three separate donors for each condition: KLF1-Empty vector control (control, open bar), KLF1-LIN28A-OE (KLF1, red bar), SPTA1-Empty vector control (control, open bar), and SPTA1-LIN28A-OE (SPTA1, blue bar). Asterisks indicate p<0.05.</p

<i>LIN28A</i> erythroid-specific over-expression does not affect cell proliferation or prevent terminal maturation of cultured erythroblasts.

<p>Cell proliferation was assessed by cell counts performed on <b>(A)</b> culture day 14 and <b>(B)</b> culture day 21. Mean fold change ± SD from three separate donors for each condition: KLF1-Empty vector control (C, open bar), KLF1-LIN28A-OE (KLF1, red bar), SPTA1-Empty vector control (C, open bar), and SPTA1-LIN28A-OE (SPTA1, blue bar). Representative flow cytometry dot plots of cells stained with antibodies against transferrin receptor (CD71) and glycophorin A (GPA) cultured on <b>(C-F)</b> day 14 and <b>(G-J)</b> day 21 with percentages shown. KLF1-Empty vector control (control, panels C and G), KLF1-LIN28A-OE (KLF1, panels D and H), SPTA1-Empty vector control (control, panels E and I), and SPTA1-LIN28A-OE (SPTA1, panels F and J).</p

Erythroid-specific <i>LIN28A</i> over-expression effects upon globin gene and protein levels in cultured adult erythroblasts.

<p><i>LIN28A</i> over-expression driven by KLF1 or SPTA1 promoter compared to control samples in the mRNA expression levels of <b>(A)</b><i>alpha</i>-, <i>mu</i>-, <i>theta</i>- and <i>zeta</i>-<i>globins</i> and <b>(B)</b><i>beta</i>-, <i>delta</i>-, <i>gamma</i>- and <i>epsilon</i>-<i>globins</i>. Analyses were performed at culture day 14. <b>(C)</b> HPLC analysis of hemoglobin from each respective control, KLF1-LIN28A-OE, and SPTA1-LIN28A-OE erythroblasts at culture day 21. KLF1-Empty vector control (C, open bar), KLF1-LIN28A-OE (KLF1, red bar), SPTA1-Empty vector control (C, open bar), and SPTA1-LIN28A-OE (SPTA1, blue bar). Representative HPLC tracing from <b>(D)</b> KLF1-Empty vector control (gray tracing), <b>(E)</b> KLF1-LIN28A-OE (red tracing), <b>(F)</b> SPTA1-Empty vector control (gray tracing), and <b>(G)</b> SPTA1-LIN28A-OE (blue tracing). Mean value ± SD three separate donors for each condition. Asterisks indicate p<0.05.</p